Fig. 3 .

PLD1 reporter assay of mock- and mutated Hras-NIH3T3 cells and effects of mithramycin A and siRNA (pSilencer) of Sp1 on PLD protein level and promoter activity.

(a) and (b) Using mock- (open column) and mutated Hras-NIH3T3 cells (solid column), promoter analyses were performed using various luciferase vectors containing human PLD1 5’ promoter shown at the left part of figure. Similar experiments using DLD-1 cells were performed (results are described in the text but data are not shown). Statistical significance was calculated by Student’s t test. (c) Mutated Hras-NIH3T3 cells were treated for 24 h with mithramycin A (100 nM or 500 nM). Western blotting was performed using anti-PLD1, anti-PLD2 and anti-Sp1 antibodies, respectively. PLD1 promoter assay was performed using –154 bp PLD1/Luc. Five hundred nM of mithramycin A was added for 24 h after reporter vector transfection. The culture was performed in triplicate. Statistical significance was calculated by Student t test. *** denotes p<0.005 (d) Similar experiments were performed using pSilencer siRNA Sp1 (siRNASp1) and its negative control pSilencer (vec). Two µg of silencer vectors were used for 1 ml culture, and the transfection was accomplished using Lipofectin. Results of Western blotting and relative promoter activity are illustrated.